This invention relates to novel genetic constructs designed to permit expression or synthesis multidomain proteins containing extended repetitive sequences, particularly those useful for the creation of molecular adjuvants and immunogens.
The complement system consists of a set of serum proteins that are important in the response of the immune system to foreign antigens. The complement system becomes activated when its primary components are cleaved and the products, alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonisation of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or a normal slow activation may be amplified in the presence of cell walls of invading organisms such as bacteria and viruses (the alternative pathway). The complement system interacts with the cellular immune system through a specific pathway involving C3, a protein central to both classical and alternative pathways. The proteolytic activation of C3 gives rise to a large fragment (C3b and exposes a chemically reactive internal thiolester linkage which can react covalently with external nucleophiles such at the cell surface proteins of invading organisms or foreign cells. As a result, the potential antigen is xe2x80x98taggedxe2x80x99 with C3b and remains attached to that protein as it undergoes further proteolysis to iC3b and C3d,g. The latter fragments are, respectively, ligands for the complement receptors CR3 and CR2. Thus the labelling of antigen by C3b can result in a targeting mechanism for cells of the immune system bearing these receptors.
That such targeting is important for augmentation of the immune response is first shown by experiments in which mice were depleted of circulating C3 and then challenged with an antigen (sheep erythrocytes). Removal of C3 reduced the antibody response to this antigen. (M. B. Pepys, J.Exp.Med, 140, 126-145, 1974). The role of C3 was confirmed by studies in animals genetically deficient in either C3 or the upstream components of the complement cascade which generate C3b, i.e. C2 and C4, (J. M. Ahearn and D. T. Fearon, Adv.Immunol. 46, 183-219, 1989). More recently, it has been shown that linear conjugation of a model antigen with more than two copies of the murine C3d fragment sequence resulted in a very large (1000-10000-fold) increase in antibody response in mice compared with unmodified antigen controls (P. W. Deropsey et al, Science, 271: 348-350, 1996; WO96/17625, PCT/GB95/02851). The increase could be produced without the use of a conventional adjuvants such as Freund""s complete adjuvant. The mechanism of this remarkable effect was demonstrated to be high-affinity binding of the multivalent C3d construct to CR2 on B-cells, followed by co-ligation of CR2 with another B-cell membrane protein, CD19 and with membrane-bound immunoglobulin to generate a signal to the B-cell nucleus.
In these experiments, the unmodified antigen control and linear fusions with one or two C3d domains were prepared by transfection of the appropriate coding plasmids into L cells followed by the selection of high-expressing clones. The most immunogenic construct that with three C3d units, had to be expressed transiently in COS cells and this procedure gave a very poor yield of the fusion protein. In part, the low yield could be attributed to the generation of species containing the antigen but with lower molecular weights, corresponding to fewer than three C3d units. It was unclear from the published work of Dempsey et al whether the latter molecules originated by proteolysis of the three-C3d construct or whether they were due to a recombination event in vivo.
Using another expression system but the same C3d constructs as Dempsey et al. we have now obtained evidence that the generation of molecules with  less than 3 C3d units from DNA encoding 3xc3x97 C3d repeats is due to loss of one or more C3d units by homologous recombination and not due to post-translational processing (see below). This observation has also identified an efficient system for the expression of the C3d monomer.
It is known generally that the production of high molecular weight polypeptides containing multiple repeating sequences is difficult because of the tendency of repeated DNA sequences to undergo rearrangement during replication. Some of the limitations on internal repetitiveness in plasmids have been discussed by Gupta (Bio/Technology 1. 602-609, 1983). Ferrari et al (U.S. Pat. No. 5,641,648) have described methods for expression of repetitive sequences using synthetic genes constructed from monomeric units which are concatenated by ligation. DNA sequences encoding the same repeated amino acid sequence but differing in nucleotide sequence either within or between monomers were constructed by exploiting the redundancy of the genetic code. The resulting lack of precise repetitiveness at the nucleotide level reduced homologous recombination to the point where the repeated oligopeptide sequence could be expressed. The work of Ferrari et al was restricted to relatively short repeating units of 4 to 30 codons (amino-acids) repeated a large number of times (typically xcx9c30-fold).
The present invention describes a general method for introducing variability into entire genes or fragments of genes, particularly those encoding autonomously folding protein domains or motifs of greater than 30 amino acids, in such a way that different DNA units encoding identical or near-identical amino acid sequences can be concatenated and expressed to give domain oligomers.
The invention comprises the following elements:
1. The construction of novel synthetic DNA sequences encoding an autonomously folding polypeptide domain and using in these DNA sequences the maximum third-base redundancy in each codon permitted by the genetic code which is consistent with a continuous reading frame and retention of the amino acid sequence. These mixtures of DNA molecules are termed xe2x80x98Fuzzy Gazesxe2x80x99.
2. Using these libraries to isolate or design concatamers in which the DNA repeats differ from each other in the third base positions. These concatamers may be made with or without in-frame coding regions for other proteins.
3. Placing these sequences either as mixed populations or single characterised concatamers into a suitable expression vector and expressing the population in a recombinant host cell.
4. The use of assays able to detect the presence of repeated-domain expressed protein products. Host cell clones are screened for those capable of producing useful levels of functionally active polypeptide concatamers/fusion proteins.
5. Where necessary, characterising one or more unique DNA sequences derived from these clones and encoding the expressed product.
6. Using the unique chemical reactivity of single cysteine residues in expressed proteins to assemble protein derivatives with multiple copies of a domain by post-translational chemical modification combined with concatamerisation at the DNA level.
In specific embodiments of the invention, the autonomously folding repeated protein domain is a ligand for one or mare cell surface receptors involved in the regulation of the immune system. One such example is human or murine C3d or C3d,g polypeptide sequence or another peptide ligand of CR2 (CD21) or CD 19.
In a second embodiment the additional domain may be an immunogen, particularly an antigenic protein or region of a protein. Examples of polypeptide immunogens include but are not restricted to: the Hepatitis B surface antigen, meningococcal surface proteins, proteins expressed at various stages of the life cycle of the malaria parasite, the glucan-binding region of streptococcal glucosyltransferases, the haemagglutinin (H) and neuraminidase (N) proteins of influenza virus strains and the D-repeat regions of the fibronectin binding proteins of staphylococci.
Optionally, an antigen oligomer may be fused to a C3d oligomer, either or both component being expressed from fuzzy or partially fuzzy genes.
In a third embodiment, the expressed oligomeric protein may be derivatised to facilitate post-translational linkage to an antigen or other protein. Preferably, such derivatisation is effected by engineering a reactive residue such as a free cysteine or a thiolester group at a unique site in the oligomer, preferably at the C- or N-terminus.
In a further aspect, the invention also provides for expression of closely related polypeptides in a single linear molecule by the ligation of fuzzy DNA sequences encoding near-identical amino acid sequences. In this context, near-identical signifies sequences differing by at least one amino acid but not in more 10% of the total number of amino acids. Examples of such constructs include but are not restricted to genes encoding several variations of a protein or antigen and concatenated immunoglobulin single-chain Fv fragments with a similar overall architecture but containing small variations in the complementarity-determining regions so that they recognise different antigens.
Another embodiment of the invention utilises the novel DNA sequences identified by the selection process noted above as components of expression vectors for genetic (or DNA) immunisation. In this application, the preferred DNA sequences for expression in a given cell type (such as a human cell line) are identified by screening cells of that type transfected with a fuzzy or partially fuzzy DNA pool (within a suitable vector) for expression of the desired construct. This application may be further extended by chemically linking the expressed, derivatised C3d (or other protein) oligomers to DNA-binding molecules such as cationic lipids, lipopeptides or liposomes so that vectors for DNA immunisation may be targeted to particular cell types. Thus, for example, a C3d trimer linked to a liposome containing DNA encoding a (C3d)3-antigen fusion could be targeted to dendritic cells. Expression of the construct by the dendritic cells could then present a targeted antigen locally to further B-lineage cells thus achieving a dual-level selectivity.
The above steps involve the following general processes:
The invention provides a process for preparing oligomeric polypeptides according to the invention which process comprises expressing DNA encoding said polypeptide in a recombinant host cell and recovering the product. That process may comprise the steps of :
(i) preparing a variable (replicable expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said polypeptide;
ii) transforming a host cell with said vector;
iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said polypeptide; and
iv) recovering said polypeptide in an active form.
The variant DNA polymers comprising a nucleotide sequence that encodes the polypeptide also forms part of the invention.
The process of the invention may be performed using conventional recombinant techniques such as described in Sambrook et al., Molecular Cloning: A laboratory manual 2nd Edition. Cold Spring Harbor Laboratory Press (1989) and DNA Cloning vols I, II and III (D. M. Glover ed., IRL Press Ltd).
The invention also provides a process for preparing the DNA polymer by the condensation of appropriate mono-, di- or oligomeric nucleotide units.
The preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate. Thus, the DNA polymer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al., in Biochemistry 1985, 24, 5090-5098.
The DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.
Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20xc2x0-70xc2x0 C., generally in a volume of 50 xcexcl or less with 0.1-10 xcexcg DNA. Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase 1 (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10-37xc2x0 C., generally in a volume of 50 xcexcl or less Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4xc2x0 C. to 3xc2x0 C., generally in a volume of 50 xcexcl or less.
The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in xe2x80x98Chemical and Enzymatic Synthesis of Gene Fragmentsxe2x80x94A Laboratory Manualxe2x80x99 (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes M. Singh, B. S. Sproat and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus and H. Koester. Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal., 1984, 3, 801. Preferably an automated DNA synthesiser (for example, Applied Biosystems 381A Synthesiser) is employed.
The DNA polymer is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the polypeptide. The DNA molecules may be obtained by the digestion with suitable restriction enzymes of vectors carrying the required coding sequences.
The precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired product. The DNA molecule encoding the polypeptide may be constructed using a variety of methods including chemical synthesis of DNA oligonucleotides, enzymatic polymerisation, restriction enzyme digestion and ligation. The design of a suitable strategy for the construction of the DNA molecule coding for the polypeptide is a routine matter for the skilled worker in the art.
The systematic variation of third-base usage is described in more detail below (Example 3, Table 1) and additional consideration may be given to the avoidance of rarely used codons of the particular host cell.
The expression of the DNA polymer encoding the polypeptide in a recombinant host cell may be carried out by means of a replicable expression vector capable, in the host cell of expressing the DNA polymer. The expression vector is novel ad also forms part of the invention.
The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the polypeptide, under ligating conditions.
The ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired. Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic, such as E. coli, mammalian, such as mouse C127, mouse myeloma. Chinese hamster ovary, or other eukaryotic (fungi e.g. filamentous fungi or unicellular yeast or an insect cell such as Drosophila or Spodopiera). The host cell may also be in a transgenic animal. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses derived from, for example, baculoviruses or vaccinia.
The DNA polymer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the fragment e.g. bovine papillomavirus vectors in mouse C127 cells, or amplified vectors in Chinese hamster ovary cells (DNA Cloning Vol. II D. M. Glover ed. IRL Press 1985; Kaufman R. J. et al., Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G. N. and Hamer, D. H. Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D. V. et al. European Patent Application No. 0093619, 1983).
The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Sambrook et al., cited above. Polymerisation and ligation may be performed as described above for the preparation of the DNA polymer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20xc2x0-70xc2x0 C., generally in a volume of 50xcexcl or less with 0.1-10 xcexcg DNA.
The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Sambrook et al., cited above, or xe2x80x9cDNA Cloningxe2x80x9d Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli, may be treated with a solution of CaCl2 (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol or by electroporation as for example described by Bio-Rad Laboratories, Richmond, Calif., USA, manufacturers of an electroporator. Eukaryotic cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells or by using cationic liposomes.
The invention also extends to a host cell transformed with a variable replicable expression vector of the invention.
Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Sambrook et al., and xe2x80x9cDNA Cloningxe2x80x9d cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45xc2x0 C.
The protein product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial such as E. coli and the protein is expressed intracellularly, it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is eukaryotic, the product is usually isolated from the nutrient medium.
Where the host cell is bacterial, such as E. coli, the product obtained from the culture may require folding for optimum functional activity. This is most likely if the protein is expressed as inclusion bodies. There are a number of aspects of the isolation and folding process that are regarded as important. In particular, the polypeptide is preferably partially purified before folding, in order to minimise formation of aggregates with contaminating proteins and minimise misfolding of the polypeptide. Thus, the removal of contaminating E. coli proteins by specifically isolating the inclusion bodies and the subsequent additional purification prior to folding are important aspects of the procedure.
The folding process is carried out in such a way as to minimise aggregation of intermediate-folded states of the polypeptide. Thus, careful consideration needs to be given to, among others, the salt type and concentration, temperature, protein concentration, redox buffet concentrations and duration of folding. The exact condition for any given polypeptide generally cannot be predicted and must be determined by experiment.
There are numerous methods available for the folding of proteins from inclusion bodies and these are known to the skilled worker in this field. The methods generally involve breaking all the disulphide bonds in the inclusion body, for example with 50 mM 2-mercaptoethanol, in the presence of a high concentration of denaturant such as 8M urea or 6M guanidine hydrochloride. The next step is to remove these agents to allow folding of the proteins to occur. Formation of the disulphide bridges requires an oxidising environment and this may be provided in a number of ways, for example by air, or by incorporating a suitable redox system, for example a mixture of reduced and oxidised glutathione.
Preferably, the inclusion body is solubilised using 8M urea, in the presence of mercaptoethanol, and protein is folded, after initial removal of contaminating proteins, by addition of cold buffer. A preferred buffer is 60 mM ethanolamine containing 1 mM reduced glutathione and 0.5 mM oxidised glutathione. Ideally, the nature of the buffer is determined experimentally in order to obtain a protein product that is functionally active. The folding is preferably carried out at a temperature in the range 1 to 5xc2x0 C. over a period of 1 to 4 days.
An alternative to the addition of cold buffer is to buffer-exchange the fully reduced protein at room temperature using Sephadex G25 Medium into, for example, 0.3M ethanolamine/1 mM EDTA/1 mM cysteine USP/2 mM L-cystine.2HCl (pH adjustment not required). The solution should be clear, or slightly cloudy -dependent on the level of impuritiesxe2x80x94and is left static approx 2-30xc2x0 C. for 1 to 4d. As previously, the exact buffer conditions and temperature should be determined experimentally for each individual protein and are not restricted to those described above.
If any precipitation or aggregation is observed, the aggregated protein can be removed in a number of ways, for example by centrifugation or by treatment with precipitants such as ammonium sulphate.
The polypeptide portion of the derivative of the invention may include a C-terminal cysteine to facilitate post-translational modification. Expression in a bacterial system is preferred for some proteins of moderate size (up to xcx9c70 kDa) and with  less than xcx9c8 disulphide bridges. More complex proteins for which a free terminal cysteine could cause refolding or stability problems may require expression in eukaryotic cells.
The use of insect cells infected with recombinant baculovirus encoding the polypeptide portion is a preferred general method for preparing more complex proteins, particularly the C3d oligomers of the invention.
A preferred method of handling proteins derivatised with cysteine is as a mixed disulphide with mercaptoethanol or glutathione or as the 2-nitro, 5-carboxyphenyl thio-derivative as generally described below.
Where the oligomeric polypeptide derivative of the invention includes a single cysteine, chemical ligation to a second polypeptide containing a unique cysteine may be employed.
The bridge is generated by conventional disulphide exchange chemistry, by activating a thiol on one polypeptide and reacting the activated thiol with a free thiol on the other polypeptide. Such activation procedures make use of disulphides which form stable thiolate anions upon cleavage of the Sxe2x80x94S linkage and include reagents such as 2,2xe2x80x2 dithiopyridine and 5,5xe2x80x2-dithiobis-(2-nitrobenzoic acid) (DTNB) that form intermediate mixed disulphides capable of further reaction with thiols to give stable disulphide linkages. One polypeptide activated in this way is then reacted with the second containing the free thiol. The precise conditions of pH, temperature, buffer and reaction time will depend on the nature of the reagent used and the polypeptide to be modified. The polypeptide linkage reaction is preferably carried out by mixing the modified polypeptides in neutral buffer in an approximately equimolar ratio. The reaction should preferably be carried out under an atmosphere of nitrogen. Preferably, UV-active products are produced (e.g. from the release of pyridine 2-thione from 2-pyridyl dithio derivatives) so that coupling can be monitored.
After the linkage reaction, the polypeptide conjugate can be isolated by a number of chromatographic procedures such as gel filtration, ion-exchange chromatography, affinity chromatography or hydrophobic interaction chromatography. These procedures may be either low pressure or high performance variants.
The conjugate may be characterised by a number of techniques including low pressure or high performance gel filtration, SDS polyacrylamide gel electrophoresis or isoelectric focussing.
In a further aspect, therefore, the invention provides a process for preparing a derivative according to the invention, this process comprises expressing DNA encoding the oligomeric polypeptide portion of said derivative in a recombinant host cell and recovering the product and thereafter post-translationally linking the polypeptide to a derivatised antigen or other polypeptide.
(i) DNA Cleavage
Cleavage of DNA by restriction endonucleases was carried out according to the manufacturer""s instructions using supplied buffers (New England Biolabs (U.K.) Ltd., Herts. or Promega Ltd., Hants, UK). Double digests were carried out simultaneously if the buffer conditions were suitable for both enzymes. Otherwise double digests were carried out sequentially where the enzyme requiring the lowest sait condition was added first to the digest. Once the digest was complete the salt concentration was altered and the second enzyme added.
(ii) DNA Ligation
Ligations were carried out using T4 DNA ligase purchased from Promega or New England Biolabs as described in Sambrook et al, (1989) Molecular Cloning; A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press.
(iii) Plasmid Isolation
Plasmids were isolated using Wizard(trademark) Plus Minipreps (Promega) or Qiex mini or midi kits and Qiagen Plasmid Maxi kit (QIAGEN, Surrey) according to the manufacturer""s instructions.
Plasmid pSG.C3d., YL encoding C3d monomer and plasmid pSG.(C3d)3. YL encoding C3d trimer were kindly provided by Professor D. T. Fearon, University of Cambridge.
(iv) DNA Fragment Isolation
DNA fragments were excised from agarose gels and DNA extracted using the QIAEX gel extraction kit or Qiaquick (QIAGEN, Surrey, UK), or GeneClean, or GeneClean Spin Kit or MERmaid Kit, or MERmaid Spin Kit (Bio 101 Inc, California USA) gel extraction kits according to the manufacturer""s instructions.
(v) Introduction of DNA Into E. coli 
Plasmids were transformed into competent E. coli BL21(DE3) or XL1-blue strains (Studier and Moffat. (1986), J. Mol. Biol. 189:113). The E. coli strains were purchased as a frozen competent cultures from Stratagene (Cambridge, UK).
(vi) DNA Sequencing
The sequences were analysed by a Perkin Elmer ABI Prism 373 DNA Sequencer. This is an electrophoretic technique using 36 cmxc3x970.2 mm 4% acrylamide gels, the fluorescently labeled DNA fragments being detected by a charge coupled device camera according to the manufactures instructions.
(vii) Production of Oligonucleotides
Oligonucleotides were purchased from Cruachem, Glasgow,UK
(viii) Generation of Baculovirus Vectors
Plasmids described in this invention having the prefix pBP (e.g. pBP68-01 described below) are used to generate baculovirus vectors and express the encoded recombinant polypeptides by the following methods (Sections (viii) to (x)). Purified plasmid DNA was used to generate recombinant baculoviruses using the kit xe2x80x98The BacPak Baculovirus Expression Systemxe2x80x99 according to the manufacturers protocols (Clontech, Calif., USA). The insect cell line Sf9 (ATCC) was grown in IPL-41 medium (Sigma, Dorset, UK) supplemented according to manufacturers recommendations with yeast extract, lipids and pluronic F68 (all from Sigma) and 1% (v/v) foetal calf serum (Gibco, Paisley, UK)xe2x80x94this is termed growth medium. Cells were transfected with the linearised baculovirus DNA (supplied in the kit) and the purified plasmid. Plaque assays (see method below) were carried out on culture supernatants and a series of ten-fold dilutions thereof to allow isolation of single plaques. Plaques were picked using glass Pasteur pipettes and transferred into 0.5 ml aliquots of growth medium. This is the primary seed stock.
(ix) Plaque Assay of Baculoviruses
1xc3x97106 Sf9 cells were seeded as monolayer cultures in 30 mm plates and left to attach for at least 30 minutes. Them medium was poured off and virus inoculum in 100 xcexcl growth medium was dripped onto the surface of the monolayer. The plates were incubated for 30 minutes at room temperature, occasionally tilting the plates to prevent the monolayer from drying out. The monolayer was overlaid with a mixture of 1 ml growth medium and 3% (w/v) xe2x80x9cSeaplaquexe2x80x9d agarose (FMC, ME) warmed to 37xc2x0 C. and gently swirled to mix in the inoculum. Once set a liquid overlay of 1 ml growth medium was applied. The plates were incubated in a humid environment for 3-5 days.
Visualisation of plaques was achieved by addition to the liquid overlay 1 ml phosphate buffered saline (PBS) containing neutral red solution at 0.1% (w/v) from a stock solution of 1% (w/v) (Sigma, Dorset, UK). Plaques were visible as circular regions devoid of stain up to 3 mm in diameter.
(x) Scale-up of Baculovirus Vectors and Protein Expression
200 xcexcl of the primary seed stock was used to infect 1xc3x97106 Sf9 monolayer cell cultures in 30 mm plates. The seed stock was dripped onto the monolayer and incubated for 20 minutes at room temperature, and then overlaid with 1 ml growth medium. The plates were incubated at 27xc2x0 C. in a humid environment for 3-5 days. The supernatant from these cultures is Passage 1 virus stock. The virus titre was determined by plaque assay and further scale up was achieved by infection of monolayer cultures or suspension cultures at a multiplicity of infection (moi) of 0.1. Virus stocks were passaged a maximum of six times to minimise the emergence of defective virus.
Expression of recombinant proteins was achieved by infection of monolayer or suspension cultures in growth medium with or without foetal calf serum (FCS). Where FCS was omitted cells conditioned to growth in the absence of FCS were used. Virus stocks between passage 1 and 6 were used to infect cultures at a moi of  greater than 5 per cell. Typically, infected cultures were harvested 72 hours post infection and recombinant proteins isolated either from the supernatants or the cells.
(xi) Selection of Stable Variants of (C3d)3 Expressed in Insect Cell Using Baculovirus Vectors
Baculovirus transfer vector plasmids encoding uncharacterised C3d concatamers containing one or more fuzzy C3d domains are transformed into competent E.coli XL1-blue strain according to Method (v). The resulting colonies each contain a single isolate which may be comprised of one or more fuzzy C3d domains and may also contain one copy of C3d with the original DNA sequence derived from pSG.C3d. YL. Individual colonies are scaled up and DNA extracted according to Method (iii) (miniprep method). Aliquots of DNA are cotransfected into insect cells with BacPak6 linear DNA using a modification of the manufacturers protocol; 2xc3x97105 Sf9 cells are seeded into flat-bottomed microtitre plates and allowed to attach for 30 minutes in serum free medium. If cells are grown in medium containing serum the monolayer is washed three times in serum free medium. The volume of medium in each well is adjusted to a standard volume (typically 50-150 xcexcl).
Each plasmid isolate 0.5 xcexcl of miniprep plasmid DNA was taken (at concentrations ranging from 0.5 to 5 xcexcg/ul) is mixed with 0.1 xcexcl to 2 xcexcl of BacPak6 DNA (Clontech) (at concentration supplied) and 5-25 xcexcl 50 xcexcg/ml of Lipofectin reagent (Life Technologies Inc.) and incubated at room temperature for 15 minutes to allow lipofectin/DNA complexes to form. The mixture is dripped onto the surface of the medium in a single microtitre well and swirled gently with the pipette tip. A single microtitre dish may contain transfections of up to 96 plasmid isolates, one well per isolate. The transfections are incubated in a humid environment at 27xc2x0 C. for five hours.
The medium containing the Lipofectin/DNA is then removed from the monolayer and replaced with 100 xcexcl medium which may contain 1% FCS unless this is considered likely to interfere with subsequent assays. The plate is incubated in a humid environment at 27xc2x0 C. for three to five days. After three days the plate is visibly inspected for evidence of baculovirus infection. When a significant number of wells show signs of infection (retarded cell growth and enlarged or dumbell-shaped cells) aliquots of culture supernatant art harvested from each well stored under sterile conditions for subsequent expansion. Further aliquots of culture supernatant or cells are subjected to biochemical or biological assay to determine the presence or absence of C3d monomers and oligomers. For example, 20 xcexcl aliquots of supernatant may be subjected to SDS-PAGE and/or Western blotting (see Methods Section (xii) and (xiv)).
Selected clones showing significant yields of (C3d)3 are scaled up from stored supernatants according to Method (x). Plasmids corresponding to the selected clones are also scaled up for large-scale plasmid DNA extraction according to Method (iii) and sequenced according to Method (vi).
(xii) pBROC413
The plasmid pT7-7 [Tabor, S (1990), Current Protocols in Molecular Biology, F. A. Ausubel, Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, (eds), pp. 16.2.1-16.2.11, Greene Publishing and Wiley-Interscience, New York.] contains DNA corresponding to nucleotides 2065-4362 of pBR322 and like pBR322 can be mobilised by a conjugative plasmid in the presence of a third plasmid ColK. A mobility protein encoded by ColK acts on the nic site at nucleotide 2254 of pBR322 initiating mobilization from this point. pT7-7 was digested with LspI and BglII and the protruding 5xe2x80x2 ends filled in with the Klenow fragment of DNA Polymerase I. The plasmid DNA fragment was purified by agarose gel electrophoresis, the blunt ends ligated together and transformed into E. coli DHI by electroporation using a Bio-Rad Gene Pulser and following the manufacturers recommended conditions. The resultant plasmid pBROC413 was identified by restriction enzyme analysis of plasmid DNA.
The deletion in pBROC413 from the LspI site immediately upstream of the f10 promoter to the BglII site at nucleotide 434 of pT7-7 deletes the DNA corresponding to nucleotides 2065-2297 of pBR322. The nic site and adjacent sequences are therefore deleted making pBROC413 non mobilizable.
(xiii) pDB1013
The construction of this plasmid, a derivative of pBROC413, is fully described in Dodd et al (1995) Protein Expression and Purification 6: 727-736.
(xiv) Protein Purification
A number of standard chromatographic techniques can be used to isolate the C3d-containing proteins, e.g. such methods as ion-exchange and hydrophobic interaction matrixes chromatography utilising the appropriate buffer systems and gradient to purify the target proteins. The properties of the C3d containing fusion polypeptides will vary depending on the nature of the fusion protein.
The C3d molecules constructed to contain the C-terminal affinity tag Glu-Glu-Phe can be purified by affinity chromatography using the YL1/2 antibody coupled to Sepharose 4B as described by Dempsey et al (WO 0617625; PCT/GB95/02851).
(xv) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE was carried out generally using the Novex system (Novex GmbH, Heidleburg) according to the manufacturer""s instructions. Pre-packed gels (4-20% acrylamide gradient, containing a Tris/glycine buffer) were usually used. Samples for electrophoresis, including protein molecular weight standards (for example LMW Kit, Pharmacia, Sweden or Novex Mark 12, Novex, Germany) were usually diluted in 1% (w/v) SDSxe2x80x94containing buffer (with or without 5% (v/v) 2-mercaptoethanol), and left at room temperature for 5 to 30 min before application to the gel.
(xvi) Immunoblotting
(a) Dot blot
Immobilon membranes (Millipore, Middlesex, UK) were activated by immersion in methanol for 20 seconds and then washed in PBS for five minutes. The membrane was placed into a vacuum manifold Dot Blotter (Bio-Rad Laboratories, Watford, UK). Crude extracts from cells or culture supernatants were transferred onto the membrane by applying a vacuum and washed through with PBS. Without allowing the membrane to dry out, the Dot Blotter was dismantled and the membrane removed.
(b) Western Blotting
Samples of cell extracts and purified proteins were separated on SDS-PAGE as described in Section (xv). The Immobilon membrane was prepared for use as in (a) above. The gel and the membrane were assembled in the Semi-Dry Transfer Cell (Trans-Blot SD, Bio-Rad Laboratories) with the Immobilon membrane towards the anode and the SDS-PAGE gel on the cathode side. Between the cathode and the gel were placed 3 sheets of Whatman 3M filter paper cut to the size of the gel pre-soaked in a solution of 192 mM 6-amino-n-caproic acid, 25 mM Tris pH 9.4 containing 10% (v/v) methanol. Between the anode and the membrane were placed two sheets of Whatman 3M filter paper cut to the size of the gel and soaked in 0.3M Tris pH 10.4 containing 10% (v/v) methanol next to the anode and on this was laid a further sheet of Whatman 3M filter paper pre-soaked in 25 mM Tris pH 10.4 containing 10% (v/v) methanol.
The whole-assembled gel assembly was constructed to ensure the exclusion of air pockets. The proteins were tranferred from the SDS-PAGE to the Immobilon membrane by passing 200 mA current through the assembly for 30 minutes.
(c) Immunoprobing of Dot Blot and Western Membranes
The membranes were blocked by incubating the membrane for 1 h at room temperature in 50 ml of 10 mM phosphate buffer pH 7.4 cotaining 150 mM NaCl, 0.02% (w/v) Ficoll 400, 0.02% (w/v) polyvinylpyrolidine and 0.1% (w/v) bovine serum albumin (BSA). The appropriate primary antibody was diluted to its working concentration in antibody diluent, 20 mM sodium phosphate buffer pH 7.4 containing 0.3M NaCl, 0.5% (v/v) Tween-80 and 1.0% (w/v) BSA. The membrane was incubated for 2 h at room temperature in 50 ml of this solution and subsequently washed three times for 2 minutes in washing buffer, 20 mM sodium phosphate pH 7.4 containing 0.3M NaCl and 0.5% (v/v) Tween-80. The membrane was then transferred to 50 ml of antibody diluent buffer containing a suitable dilution of the species specific antibody labelled with the appropriate label, e.g. biotin, horse radish peroxidase (HRP), for the development process chosen and incubated for 2 h at room temperature. The membrane was then washed in washing buffer as described above. Finally, the blot was developed according to the manufacturer""s instructions.
The appropriate dilution of antibody for both the primary and secondary antibodies refers to the dilution that minimises unwanted background noise without affecting detection of the chosen antigen using the development system chosen. This dilution is determined empirically for each antibody.
(xvii) Measurement of Biological Activity
The biological function of C3d monomer produced in baculovirus can he tested for its ability to hind to its receptor, complement receptor-2 (CR2). As C3d is a product of the process of complement activation and subsequent degradation of C3b by the serum protease Factor I, it was also of interest to test C3d for its possible effect on complement activation using a classical pathway haemolytic assay.
(a) Competitive Binding Assay of C3d to Raji Cells
In this assay the ability of a new construct expressing at least one unit of C3d to compete with a control 125I-HEL-C3d for CR2 binding sites on the surface of Raji cells, a B-lymphoblastoid cell line, is assessed. Raji cells, 5xc3x97107 to 7xc3x97107 cells/ml are incubated for 1 h at 4xc2x0 C. with 1 nM 125I-HEL-C3d and incremental concentrations of C3d containing molecule of interest. The cells are then centrifuged through a dibutyl-diiso-octyl-phthalate cushion, and the amount of radioactivity associated with the pellet determined. From this data the amount of the C3d-containing protein under test necessary to produce a 50% reduction in 1 mM 125I-HEL-C3d can be determined.
(b) Competitive ELISA demonstrating C3d Binding to CR2
C3d is used to coat 96-well microtitre plates at 5 xcexcg/ml in O. 1M NaHCO3 by incubating overnight at 4xc2x0 C. The plates are then blocked in 1% BSA 0.1% Tween-20 in PBS for 1 h at room temperature subsequently they are incubated with either 25 xcexcl/well of C3d or from the C3d containing molecule under test in a dose response manner. To the wells is added 25 xcexcl/well of a sub-saturating concentration of co-ligated CR2. IgGl at 125 pM and incubated for 1 h at room temperature. The plates are then washed three times with PBS containing 0.1% (v/v) Tween-20. The amount of CR2 bound to the immobilised C3d is detected using a 1:3000 dilution of HRP-labeled goat anti-mouse-IgGl-antibody in PBS containing 0.1% (v/v)Tween-20 and incubated for 1 h at room temperature the wells were then washed as described above. The presence of HRP-antibody is detected using the TMB Peroxidase EIA Substrate Kit according to the manufacturer""s instructions (Bio-Rad, UK).
(c) Anti-complement Activity Measured by the Haemolysis of Sheep Erythrocytes
Measuring the inhibition of complement-mediated lysis of sheep erythrocytes sensitised with rabbit antibodies (Diamedix Corporation, Florida, USA) assesses functional activity of complement inhibitors. Human serum diluted 1:125 or 1:100 in 0.1M Hepes/0.15 M NaCl/0.1% gelatin pH 7.4 was used as a source of complement. The serum was pooled from volunteer blood donations essentially as described in J. V. Dacie and S. M. Lewis. Practical Haematology. Churchill Livingstone, Edinburgh. 1975. Briefly, blood (10-20 ml) was warmed to 37xc2x0 C. for 5 minutes, the clot removed and the remaining serum clarified by centrifugation. The serum fraction was split into small aliquots and stored at xe2x88x92196xc2x0 C. Aliquots were thawed as required and diluted in the Hepes buffer immediately before use.
Inhibition of complement-mediated lysis of sensitised sheep erythrocytes is measured using a standard haemolytic assay using a v-bottom microtitre plate format as follows:
50 xcexcl of a range of concentrations of inhibitor (typically in the region of 0.1-100 nM) diluted in Hepes buffer are mixed with 50 xcexcl of the diluted serum and 100 xcexcl of sensitised sheep erythrocytes and then incubated for 1 hour at 37xc2x0 C. Samples are spun at 1600 rpm at ambient temperature for 3 minutes before transferring 150 xcexcl of supernatant to flat bottom microtitre plates and determining the absorption at 410 nm. Maximum lysis (Amax) is determined by incubating serum with erythrocytes in the absence of any inhibitor. Background lysis (Ao) is determined by incubating erythrocytes in the absence of any serum or inhibitor to check whether the inhibitor itself had any effect on lysis, erythrocytes were incubated with inhibitor alone; none of the compounds bad any direct effect on lysis of the erythrocytes. Inhibition is expressed as a fraction of the total cell lysis such that IH50 represents the concentration of inhibitor required to give 50% inhibition of lysis.   IH  =            A      -      Ao              A      ⁢              xe2x80x83            ⁢              max        ·        Ao            
where 0 is equivalent to complete inhibition and 1 equals no inhibition.
(xviii) Reduction of Disulphides and Modification of Thiols in Proteins
There are a number of methods used for achieving the title goals. The reasons it may be necessary to carry out selective reduction of disulphides is that during the isolation and purification of multi-thiol proteins, in particular during refolding of fully denatured multi-thiol proteins, inappropriate disulphide pairing can occur. In addition even if correct disulphide paring does occur, it is possible that a free cysteine in the protein may become blocked, for example with glutathione. These derivatives are generally quite stable. In order to make them more reactive, for example for subsequent conjugation to another functional group, they need to be selectively reduced, with for example dithiothreitol (DTT) or with Tris (2carboxyethyl) phosphine.HCl (TCEP) then optionally modified with a function which is moderately unstable. An example of the latter is Ellman""s reagent (DTNB) which gives a mixed disulphide. In the case where treatment with DTNB is omitted, careful attention to experimental design is necessary to ensure that dimerisation of the free thiol-containing protein is minimised. Reference to the term xe2x80x98selectively reducedxe2x80x99 above means that reaction conditions e.g. duration, temperature, molar ratios of reactants have to be carefully controlled so that disulphide bridges within the natural architecture of the protein are not reduced. All the reagents are commercially available e.g. from Sigma (Poole, Dorset) or Pierce and Warriner (Chester, Cheshire).
The following general examples illustrate the type of conditions that may be used and that are useful for the generation of free thiols and their optional modification. The specific reaction conditions to achieve optimal thiol reduction and/or modification are ideally determined for each protein batch.
TCEP may be prepared as a 20 mM solution in 50 mM HEPES (approx. pH 4.5) and may be stored at xe2x88x9240xc2x0 C. DTT may be prepared at 10 mM in sodium phosphate pH 7.0 and may be stored at xe2x88x9240xc2x0 C. DTNB may be prepared at 10 mM in sodium phosphate pH 7.0 and may be stored at xe2x88x9240xc2x0 C. All of the above reagents are typically used at molar equivalence or molar excess, The precise concentrations ideally identified experimentally. The duration and the temperature of the reaction are similarly determined experimentally. Generally the duration would be in the range 1 to 24 hours and the temperature would be in the range 2 to 30xc2x0 C. Excess reagent may be conveniently removed by buffer exchange, for example using Sephadex G25. A suitable buffer is 0.1M sodium phosphate pH 7.0